Systems and methods for determining a complete blood count and a white blood cell differential count

ABSTRACT

Systems and methods analyzing body fluids such as blood and bone marrow are disclosed. The systems and methods may utilize an improved technique for applying a monolayer of cells to a slide to generate a substantially uniform distribution of cells on the slide. Additionally aspects of the invention also relate to systems and methods for utilizing multi color microscopy for improving the quality of images captured by a light receiving device.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 12/430,885, filed on Apr. 27, 2009 (now U.S. Pat. No. 9,017,610),which claims priority under 35 U.S.C. § 119 to U.S. ProvisionalApplication No. 61/047,920, filed on Apr. 25, 2008. Further, the presentapplication expressly incorporates herein by reference the applicationentitled “SYSTEMS AND METHODS FOR ANALYZING BODY FLUIDS,” U.S.Provisional Application No. 61/173,186, which was filed on Apr. 27, 2009by the same inventors as the present application. The contents of eachof these applications are expressly incorporated herein by reference intheir entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under NIH Grant NumberHL077033. The Government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to a system and process for determiningcomposition and components of fluids. More specifically the presentinvention provides improved techniques for viewing cellular morphology,and determining the number of a particular type of cell in a portion ofa body fluid.

BACKGROUND OF THE INVENTION

Pathology is a field of medicine where medical professionals determinethe presence, or absence of disease by methods that include themorphologic examination of individual cells that have been collected,fixed or air-dried, and then visualized by a stain that highlightsfeatures of both the nucleus and the cytoplasm. The collection of thecells often involves capturing a portion of a person's body fluid,placing the body fluid on a slide, and viewing the fluid on the slideusing a microscope.

One of the most commonly performed pathologic studies is the CBC (theComplete Blood Count). To perform a CBC, a sample of blood is extractedfrom a patient and then the cells are counted by automated or manualmethods. The CBC is commonly performed by using an instrument, based onthe principal of flow cytometry, which customarily aspiratesanticoagulated whole blood and divides it into several analysis streams.Using the flow cytometer a number of primary and derived measurementscan be determined including: i) red blood cell (RBC) count, hemoglobin(Hb), hematocrit (Hct), red blood cell indices (mean corpuscular volume,MCV, mean corpuscular hemoglobin, MCH and mean corpuscular hemoglobinconcentration MCHC), red blood cell distribution width, enumeration ofother red blood cells including reticulocytes and nucleated red bloodcells, and red blood cell morphology; ii) white blood cell (WBC) countand WBC “differential” count (enumeration of the different normal whiteblood cell types, including neutrophils, lymphocytes, eosinophils,basophils and monocytes, and the probable presence of other normal andabnormal types of WBC that are present in various disease conditions);and iii) platelet count, platelet distribution widths and other featuresof platelets including morphological features. In flow cytometers, redblood cell, WBC, and platelet morphological characterizations aretypically made indirectly, based on light absorption and lightscattering techniques and/or cytochemically based measurements. Someadvanced flow cytometers calculate secondary and tertiary measurementsfrom the primary measurements.

Flow based CBC instruments generally require extensive calibration andcontrol, maintenance, and skilled operators, and they have substantialcosts associated with acquisition, service, reagents, consumables anddisposables. One significant problem with these systems in routine useis that a large proportion of blood specimens require further testing tocomplete the assessment of the morphologic components of the CBC. Thisinvolves placing a sample of blood on a slide, smearing the sampleagainst the slide to form a wedge smear, and placing the slide under amicroscope. This process is often done manually by skilled medicaltechnologists, which increases the cost and time to receive results fromthe tests. The direct visualization of blood cells on a glass slide mustbe performed whenever the results of the automated test require furtherexamination of the blood sample. For example, a “manual” differentialcount is performed by direct visualization of the cells by anexperienced observer whenever nucleated immature RBCs are found or WBCssuspicious for infection, leukemias or other hematologic diseases arefound.

The proportion of these specimens requiring further review generallyranges from 10% to 50%, depending on the laboratory policy, patientpopulation and “flagging” criteria, with a median rate of around 27%.The most frequent reasons for retesting include the presence ofincreased or decreased number of WBCs, RBCs or platelets, abnormal celltypes or cell morphology, clinical or other suspicion of viral orbacterial infections.

In addition to additional work involved in performing manualdifferential counts, this process has a number of additional technicallimitations. These include distortions of cell morphology because ofmechanical forces involved in smearing the cells onto the slide, andcells overlapping one another, which makes visualization of individualcell morphology difficult.

SUMMARY OF THE INVENTION

The present invention provides systems and methods for placing cells ona slide. Additionally systems and method for imaging the cells areprovided. The images may be later used to perform tests such as acomplete blood count including image-based counting and assessment ofthe morphology of the formed elements of blood, including RBCs, WBCs,and platelets. Embodiments of the present invention may improve theaccuracy of the CBC by providing improved visualization of the formedelements of blood. Aspects of the present invention may analyze anddetermine the presence of certain cell types, such as abnormal orimmature WBCs that are found in cases of abnormal bone marrow functionincluding hematological malignancies. Further, the configurations of thepresent invention may decrease costs associated with instrumentation,decrease costs of consumables and reagents, require less operator timeand reagents, fewer repeated tests, and fewer moving parts than otherprior art techniques. Configurations of the present invention may alsoreduce the turn around time for many of the CBC tests that currentlyrequire visualization of blood cells after the instrumental portion ofthe test is completed, by allowing cells to be visualized on a monitorinstead of under a microscope.

Aspects of the present invention are effective at preserving cellmorphology. This may be important for patients with hematologicalmalignancies such as chronic lymphocytic leukemia (CLL) or acute myeloidleukemia (AML). The systems and methods relating to applying a monolayerof cells to a slide may enable detection of a larger number ofmorphologically well-preserved blast cells and other immature or fragilecells. This would allow their more accurate recognition at an earlierstage of the leukemic or other disease process. Certain aspects of thepresent invention provide for preparing a substantially uniformdistribution of cells across a test area of a slide.

Aspects of the present invention also relate to collecting cells in afluid (such as blood) from organic tissue, possibly mixing the cellscontained in the fluid with a diluent, collecting a sub-sample (aliquot)of a known volume from the solution, and then depositing the aliquotonto a substratum such as a slide using a dispensing device orapplicator. The cells may be allowed to air dry or may be fixed (using afixative solution) or both, depending on the examination that isanticipated. The cells may also be stained. The stained cells on thesubstratum may be counted and examined by an automated imaging systemutilizing a computer or viewed by manual microscopic examination.Digital images may be shown on a computer display to reduce the need formanual microscopic review.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: is a perspective, schematic view of a system for analyzing bodyfluids.

FIG. 1B: is a perspective, schematic view of a system for analyzing bodyfluids.

FIG. 2: is a perspective view of a slide and slide holder.

FIG. 3: is an enlarged top view of the slide and slide specimen.

FIG. 4: is alternate embodiment of the top view of the slide and slidespecimen.

FIG. 5: is a graph illustrating the correlation between SYSMEX® brandhematology analyzer (hereinafter “Sysmex”) RBC counts and the RBC countsgenerated using an embodiment of the invention.

FIG. 6: is a graph illustrating the correlation between Sysmex WBCcounts and the WBC counts generated using an embodiment of the instantinvention.

FIG. 7A: is a process flow schematic of the embodiment shown in FIG. 1A.

FIG. 7B: is a process flow schematic of the embodiment shown in FIG. 1B.

FIG. 8 is an image of rows of a blood sample distributed on a slide. Theimage was created by tiling approximately 450 individual images togetherwhile reducing overall image size.

FIG. 9 is an image of blood cells distributed on a slide acquired by adigital camera for subsequent analysis.

FIG. 10 is a flow chart of process steps carried out by the systemsdescribed herein.

DETAILED DESCRIPTION OF THE INVENTION

With reference to FIG. 1A, a system 10 for analyzing body fluids isdisclosed. The system may comprise a platform 100, a light receivingdevice 200, a computer 300, an applicator 400, a gas circulation device500, a light source 600, a dispenser 800, a discharge device 900, aslide labeler 1000, and slide label reader 1100. The following sectionsbelow include capitalized headings which are intended to facilitatenavigation through the specification. The headings are not intended tobe limiting of the invention in any manner.

The Platform 100

In embodiments which feature a platform 100, an advancer 110 may beconfigured to receive one or more slide apparatuses 700-700″. Theadvancer 110 may be attached to a surface, such as the top surface 101,of the platform. The advancer 110 may take the form of a belt as shownin FIG. 1A, the system may use a mechanical arm, gravity, magnetism,hydraulics, gears, or other locomotion techniques to move the slideapparatus along the surface 101 of the platform.

The platform 100 may also comprise a feeder 102 and a collector 106 forrespectively feeding and collecting the slide apparatuses 700 from or toa stack or rack. The feeder 102 may be equipped with a feeder propulsionmechanism 103 (such as rubberized wheels) for pushing the slides down aramp 104 onto the advancer 110. (Of course embodiments of the inventioncould be built without a ramp. For example, if the feeder is level withadvancer 110, no ramp would be needed. Alternatively, a mechanical armcould be used to grab the slide apparatus 700 and place the slideapparatus 700 on the advancer directly.) Alternate mechanisms to propelthe slide out of the feeder 102 may be used such as magnets orhydraulics. The feeder may comprise a sensor for determining how manyslides are present. The sensor could measure the weight of the slideapparatuses 700 for example to determine how many slide apparatuses werepresent. FIG. 1A illustrates 3 slide apparatuses 700 stored in thefeeder 102. The collector 106 may also comprise a sensor for determininghow many slides are present in the collector 106. The sensor may informthe computer when a preset number of slides have been analyzed or mayinform the computer of the receipt of a slide on an ongoing basis.

The Light Receiving Device 200

The light receiving device 200 may be a microscope (such as brightfieldmicroscope), a video camera, a still camera, or other optical devicewhich receives light. In embodiments using a standard brightfieldmicroscope, one containing an automated stage (a slide mover 201) andfocus may be selected. In one embodiment, a microscope may be attachedto a motorized stage and a focus motor attachment. The microscope mayhave a motorized nosepiece, for allowing different magnification lensesto be selected under computer 300 control. A filter wheel may allow thecomputer 300 to automatically select narrow band color filters in thelight path. LED illumination may be substituted for the filters, and useof LEDs may reduce the image acquisition time as compared to the timerequired for filter wheel rotation. A 1600×1200 pixel firewire cameramay be used to acquire the narrow band images.

In some cases, the light receiving device will receive light reflectedoff slide apparatus 700″ and store an image of that light. In someembodiments fluorescent emission from the cellular objects may bedetected in the light receiving device 200. However, since the lightemission source 600 can be positioned below the platform, the lightemission source may direct light so that it passes through the platform100 and the slide 701 into the light receiving device 200. The lightreceiving device may be connected to a computer through a link 11, andmay be capable of X, Y, and Z axial movement (in other embodiments amotorized stage or slide mover 201 may provide X, Y, and Z movement.)The light receiving device may comprise a link 11 such as a wire asshown in FIG. 1A, or other wireless systems may be used. The lightreceiving device 200 and any of the other components may be interfacedwith the computer 300 through a link (11-15) which may provide energy tothe component, provide instructions from the computer 300 to thecomponent, or allow the component to send information to the computer300. Light receiving device 200 may contain pan, tilt, or locomotiveactuators to allow the computer 300 to position the device 200 in anappropriate position. The light receiving device may contain a lens 210which focuses the light. The light receiving device may capture blackand white or color images. Alternatively, two or more light receivingdevices could be used to divide the processing time associated withcapturing the images. For example a low magnification image stationcould be followed by a high magnification image station. Similarly, insome embodiments, the system 10, platform 100, computer 300, or lightreceiving device 200 may direct a slide mover 201 to move the slideapparatus 700 in order to store images of all the cells in the slide.Using a slide mover 201 may be desirable, if for example, the field sizeof the light receiving device 200 is smaller than the specimen zone 710(FIG. 3).

The Computer 300

The computer 300 may be a laptop as shown in FIG. 1A, or a server,workstation, or any other type of computing device. The computer maycomprise a processor, a display 320, and interface 310, and internalmemory and/or a disk drive. The computer 300 may also comprise softwarestored in the memory or on computer readable media such as an opticaldrive. The software may comprise instructions for causing the computerto operate the light receiving device 200, the applicator 400, theapplicator controller 490, the fan 500, the platform 100, advancer 110,light source 600, dispenser 450 or 800, or any component connected toone of these components. Similarly, the computer may receive informationfrom any of these components. For example, the software may control therate of dispersal of slides from the feeder 102, and feeder 102 mayinform the computer about the number of slides present. In addition, thecomputer 300 may also be responsible for performing the analysis of theimages captured by the light receiving device. Through the analysisprocess, the computer may be able to calculate the number of a specifictype of cell in a particular volume of blood, for example for blood, redcell, white cell, and platelet counts and other measured and derivedcomponents of the CBC such as: hemoglobin content, red blood cellmorphology, or WBC differential could be calculated. The image analysissoftware may analyze each individual field and sum the total red andwhite cell counts. To calculate the total counts per microliter in thepatient vial, the number counted on the slide is multiplied by thedilution ratio and volume of the sub-sample. Results of the counts,morphologic measurements, and images of RBCs and WBCs from the slide maybe shown on the display 320. In some embodiments, the computer 300 maybe able to display numerical data, cell population histograms,scatterplots, and direct assessments of cellular morphology using imagesof blood cells displayed on the monitor. The ability to display cellularmorphology provides users of the system 10, the ability to quicklyestablish the presence or absence of abnormalities in cell morphologythat may warrant preparing an additional slide for manual review by anexperienced technician or other professional. The software may providethe computer instructions to display images 331 received from the lightreceiving device or may cause the display 330 to show the results 332(in perhaps a chart or graph for example) of an analysis of the images.Similarly, the computer 300 may be able to enumerate the number of cellsof a specific type in a particular blood volume or enumerate the numberof damaged cells, cancerous cells, or lysed cells in a particular volumeof blood. The memory of the computer may contain software to allow thecomputer to perform the analysis process. The computer may use one ormore magnifications during the analysis.

Although shown as one component, computer 300 may comprise multiplecomputers and a first computer could be used for controlling thecomponents and a second computer could be used for processing the imagesfrom the light receiving device 200. In some embodiments, the variouscomputer may be linked together to allow the computer to shareinformation. The computer 300 may also be connected to a network orlaboratory information system to allow the computer to send and receiveinformation to other computers.

The Applicator 400

The applicator station includes 1) a precision automated dispenser tipthat allows very small volumes of fluids to be handled and 2) automatedmovement of a substrate under the tip to allow the volume of fluid,e.g., diluted blood, to be dispensed over a defined area of thesubstrate.

In certain embodiments, the applicator 400 may comprise a syringe, amanual or motor driven pipettor or using a motor controlled pumpattached through a tube to a pipette tip. While many different types ofpipettes or syringes could be used, test results have shown improvedresults can be obtained through using an applicator 400 having betterthan 2% accuracy. The pump may be a peristaltic pump, a syringe pump, orother similar device that allows small volumes to be aspirated anddispensed through an orifice. Typically such an orifice will becontained in a tip 405 that is two to five millimeters in outsidediameter with an inner diameter of 0.5 millimeters. The tip 405 may bedisposable or washable. The tip 405 may be rounded to facilitateinsertion and cleaning of the tip. Fluid flow through the tip iscontrolled to allow a thin layer of blood or diluted blood to bedeposited onto the slide. By optimizing flow rate through the tip andthe relative speed and height of the tip over the slide an appropriatedensity of cells can be deposited onto the slide. Each of these factorsinfluences the other, so the proper combination of height, flow ratethrough the tip, and speed over the slide must be determined. In oneembodiment the flow rate through the tip is 0.1 microliters per secondwhile the tip is moving at a speed of 30 millimeters per second over theslide surface at a height of about 70 microns.

In use, the applicator 400 may comprise a known volume of body fluidsuch as 30 microliters (ul). The applicator may mix this fluid with astain or diluent, and eject a portion of this fluid onto the slideapparatus 700 (particularly the specimen zone 710, FIG. 3). A typicalsub-sample would be an aliquot of approximately ½ μl to 2 μl, but may bein the range of 1/10 to 10 μl. In some embodiments, the system 10 orapplicator 400 may contain a first reservoir 420 for storing the bodyfluid and a second reservoir 430 for storing diluent. In someembodiments the body fluid will not be diluted.

The system 10 or applicator 400 may contain one or more dispensers 800.The dispenser 800 (or 450 in FIG. 1B) may be used to direct a fixativeor a stain onto the slide 701. In this embodiment, the applicator 400may contain one or more fluid chambers 410 to eject body fluid, diluent,stain, and fixative from the applicator 400. Some dispensers may be ableto store both fluids and direct them sequentially onto the slide, or inalternate embodiments, two dispensers may be used (one for the fixativeand one for the stain.) Excess stain and fixative may be removed fromthe slide, by tilting the slide apparatus so that it is orthogonal (orangled) to the platform surface 101. A slide tilter 801 may be used forthis purpose. Slide filter may comprise a simple wedge as shown, or maycomprise a mechanical arm to tilt the slide.

In the embodiment shown in FIG. 1A, the stain dispenser is attached tothe platform 100. Examples of stains compatible with embodiment shown inFIG. 1A may include: Wright-Giemsa stain, Geimsa stains, and Romanowskystains. Other solutions that could be dispensed are fixatives (methanol)and buffer solutions. Other visualization methods involvingimmunocytochemical reagents or other markers of specific cell componentsmay also be used. The stain dispenser may also be embodied as a stainreservoir 450 and attached to the applicator 400 (see FIG. 1B). Examplesof stains compatible with the embodiment shown in FIG. 1B may include:Romanowsky stains, reticulocyte stains, and stains using specificantibodies. In the embodiment having dispenser 800, the dispenser candispense stain onto the slide apparatus (particularly the specimen zone710.) Dispenser 800 may take the form of a peristaltic pump. In theembodiment having a stain reservoir 450, the stain may be mixed in withthe body fluid and the diluent from reservoirs 420 and 430. The bodyfluid and the diluent may be mixed together by a mixer 440, which canmix the fluid and diluent in certain ratios. In an alternate embodiment,the slide could be immersed into one or more baths of the fixation andstaining solutions. In another embodiment, fixation and stainingsolutions could be moved across the slide using capillary action.

Various fixatives and diluents may be used with the present invention.For example 85% methanol can be used as the fixative. For some stains anethyl alcohol or formaldehyde based fixative might be used. Diluentsuseful for diluting whole blood for example, may include salt solutionsor protein solutions. Salt solutions range from “physiological saline”(0.9N), to complex mixtures of salts, to the commercial preparationPLASMALYTE® that simulates virtually all the salts found in human bloodserum. Protein solutions can range from simple solutions of bovinealbumin to PLASMANATE®, a commercial preparation with selected humanplasma proteins. Such preparations can vary in protein concentrations,buffers, pH, osmolarity, osmalality, buffering capacity, and additivesof various types. Synthetic or “substitute” versions of these solutionsmay also be usable, including FICOLL® or Dextran or otherpolysaccharides. Other substitutes may be used. An example of a diluentis PLASMALYTE® plus PLASMANATE® in the proportion of 4:1(PLASMALYTE®:PLASMANATE®). Another example of a diluent is 5% albumin.When analyzing whole blood, a dilution of 2 parts blood to 1 partdiluent can be used, where the diluent is a physiologically compatiblesolution, but a range of dilution from 0:1 (no dilution) to 10:1(diluent:blood) may be used in alternate embodiments.

The applicator may comprise a hydraulic piston for pushing the fluid outof fluid chamber 410 (like a syringe or a pipette). A tip 405 may beprovided for adjusting the flow rate of the fluid. While size of the tipdoes not affect the speed (μl/sec) in which the solution flows out ofthe tip, generally, the smaller the opening in the tip, the greater theforce (μg*distance/seconds²). Additionally, the size of the tip affectsthickness of the fluid flows 750 shown in FIGS. 2 and 3. A tip having a0.3 millimeter inner diameter may provide for a flow rate of 0.1microliters per second, and the distance from a middle point 751 of thefirst flow to the middle point 752 of the second flow may be 500microns. In order to create the flows 750 shown in FIGS. 2 and 3, thesystem 10 may be configured to account for the variances in the numberof cells in a given blood specimen. For human peripheral blood samples,the range is large but within one order of magnitude. In order toaccurately count the blood cells, the overlap between red blood cellsshould be minimized. One method to provide minimal overlapping betweencells is to lay down non-touching rows of cells from the tip of theapplicator. Increasing viscosity of the diluted fluid or the type oramount of diluent may affect the width of the final settlement positionsof the flows 750. By selecting a distance between rows to allow for thetypical variation in blood samples, all cells can be counted in allsamples. For many samples these gaps will be seen between the flows;however this does not affect the image analysis and the row and gapeffect tends not to be noticed during high magnification manual reviewunder the microscope. To avoid these gaps, a light receiving devicecould be attached to the applicator or positioned near station A (seeFIG. 7A) to allow the computer 300 to determine the width of the firstflow 751 (FIG. 3) formed by directing the cells onto the slide. Bydetermining the width of the flow, i.e. how far the blood flows sidewaysfrom location the fluid was placed on the slide, the computer 300 couldcause the applicator to adjust the gap size of the flows. The computer300 calculate the distance the second flow 752 (FIG. 3) needs to be fromthe first flow 751, and place the flows so that they settle adjacent toone another minimizing the formation of any gaps between the flows.Using this process, a gapless or contiguous flow of cells can be appliedto the specimen zone 710.

To physically place the cells on the slide 701, the computer 300 coulddirect the applicator controller 490 to perform the body fluidapplication process 7B (see FIG. 7B) which involves moving the bodyfluid chamber 410 in the X, Y, or Z directions to position the tip 405so that it tracing the eventual locations of the flows 750. In someembodiments, the X, Y, and Z directions are all perpendicular to eachother affording the applicator the ability to move in any direction in athree dimensional coordinate system.

The computer 300 may be connected to the applicator controller 490 tocontrol this movement. In the embodiment shown in FIG. 3, the controllermay position the tip at the top left corner of the specimen zone 710 andproceed to place fluid sample onto the cells by ejecting the fluid fromthe fluid chamber 410. While the ejection is occurring, the controller490 may move the tip in the positive X direction to the top rightportion of the specimen zone 710 (see FIG. 3). Once the top rightsection is reached, the controller 490 may move the tip in the negativeY direction one flow width. The flow 750 width may range from 300 to1000 microns, and flow thickness increases as the flow rate of fluid outof the tip increases and/or the speed of the tip across the slidedecreases. Additionally the viscosity of the fluid and diluent choicemay affect the width of the flow 750 (FIG. 3). Typically, the cells ofthe fluid will settle within a few seconds once placed on the slide.Once the tip has been moved one flow width, the controller may move thetip in the negative X direction to the leftmost side of the specimenzone 710. Once the leftmost side is reached, the tip again may be movedone flow width in the negative Y direction. This process may be repeateduntil the entire specimen zone is covered. In alternate embodiments, thediluted body fluid could be applied to slide with a fixed applicator andslide which moves via the moveable slide controller 760 (thisapplication process 7A is shown on FIG. 7A.) The slide controller 760may be moveable in the X, Y, Z direction to move the slide apparatus insimilar positions to allow the applicator to place flows 750 of bodyfluid on the specimen zone 710.

The number of cells placed on the slide 701 using this method will varydepending on the type of fluid being examined and the dilution ratio.Assuming whole blood were being analyzed with a 1:3 (blood:diluentratio), about 900,000 red blood cells, 45,000 platelets, and 1,000 whiteblood cells would be placed on the slide. Though FIG. 3 shows thegeneration of a uniformly distributed fluid specimen in a rectangularshape, other shapes may be constructed in a similar manner. FIG. 4,shows for example, a fluid flow comprising a plurality of concentriccircles. Like FIG. 3, the fluid flows 750 are placed adjacent to oneanother to create a uniform viewing field. This process provides ahighly uniform distribution of cells across the specimen zone 710,facilitating the analysis process. Additionally, the computer 300 canalter the appearance and width of the fluid on the zone 710. Forexample, the computer 300 may control the speed at which the tip movesacross the specimen zone, which would affect the thickness of the fluidresting on the zone. In some embodiments, speeds of 10 to 100 mm/s maybe selected in order to provide the zone with a specimen which is aboutone cell thick. The controller 490 also may select the height of the tipabove the slide 700. A height of 70+/−40 microns above the slide may beused in order to minimize damage to fluid cells when they come intocontact with the slide apparatus 700, and to maintain fluid flow fromthe tip to the substrate.

The Gas Movement Device 500

Gas movement device 500 may comprise a fan (such as shown in FIG. 1) ormay comprise other gas movement devices such as a compressor or a bellowfor example. Gas movement device 500 may be connected directly to thecomputer 300 or may be connected through another component such as theplatform 100 or the applicator 400 (as shown.) The gas movement devicepushes gas (in some cases atmospheric air) across the slide to controlthe rate at which the slide dries. Moving too much air too quickly (i.e.too high of a fan speed) across the slide can cause cells in thespecimen to burst due to too rapid drying, and too little air too slowly(i.e. too low of a fan speed) across the slide can cause the cells todry too slowly and appear to shrink. The computer 300 may select theamount of air that moves across the slide in a period of time (i.e. thecubic feet of air per second) based upon the distance the gas movementdevice is from the slide, the type of fluid being analyzed, the width ofthe flows, and averages thickness of the flows (this would be the amountcells in each flow in the Z direction). The gas movement device 500 maybe placed near the slide apparatus 700, and positioned so that thedevice directs gas so that the gas strikes the slide at an angle of30-60° angle (45° degrees can be used) for a period of about 15 to 20seconds. In some embodiments, the computer can control of humidity andtemperature settings in the vicinity of the system to allow the dryingprocess to occur without the use of a gas movement device 500.

The Light Emission Device 600

Two different embodiments of light emission device 600 are illustrated.In FIG. 1A, light emission device 600 comprises a housing 601, amultispectrum light source 610, a number of light filters 620, 620′, and620″, and a filter selector 621. As shown in FIG. 1A, a portion of thehousing has been removed to better show the light source 610. Lightsource 610 may comprise a white light source or other multispectrumlight source such as a halogen bulb, florescent bulb, or incandescentbulb etc. Filters 620-620″ may be used to filter the multispectrum lightinto a single wavelength or a narrow band of wavelengths. The filterselector 621 may select which filters appear in front of the lightsource 610. In some embodiments more than one filter may be used toallow a particular range of light to illuminate the slide. Filterselector 621, may comprise a rotation motor and a rod to spin thefilters in and out of the path of the light. In a second embodimentlight source may comprise one or more lasers or LEDs (630) which emit anarrow band of light (see FIG. 1B). An advantage for using LEDs in thissystem 10, is that LEDs can rapidly be switched on and off, allowing thelight receiving device a single black and white camera to acquire themultiple spectral images in a very short time. LEDs also produce narrowbandwidths of illumination, typically from 15 to 30 nm full width athalf maximum (the breadth of the wavelength intensity distribution athalf of the peak brightness of the maximum intensity). Also, LEDs in thevisible range do not project heat-producing infrared energy into theoptical system and are relatively long lived as compared to conventionallamps. An advantage of using narrow-band illumination rather thanunfiltered white light (i.e. broad-band illumination) is using narrowband illumination increases the sharpness of the images generated by thelight receiving device 200. If the light receiving device 200 contains alens, the presence of the lens may cause some chromatic aberration thatresult in slight focus shifts or image quality degradation when usingdifferent colors. With white light illumination this can result in anoverall degradation of the image quality. The light receiving device 200may capture a black and white image for each narrow-band ofillumination. The computer 300 may be able to correct focus and imagequality for each wavelength by adjusting the focal distance or thedistance of the lens from the slide. In some embodiments, the computer300 may shift the focus position of the lens while a number of lightcolors are emitted sequentially to improve the quality of the image.

Various wavelengths of light may be directed by the light emissiondevice 600. Two-eight or more different wavelengths of light may bedirected at the slide apparatus 700. Wavelengths such as 430 nm areuseful for imaging a hemoglobin-only image for assessing RBC morphologyand hemoglobin content. Using an image taken with such a wavelengthwhich is designed to show only red blood cells, it may also show redblood cells which are touching white blood cells. The touching red bloodcells may be digitally removed from images to make it easier for thecomputer to detect the white blood cell borders in order to make moreaccurate cellular measurements and enumeration. Light emitted at 570 nmmay be useful to provide high contrast images for platelets and nuclei.Other wavelengths may be chosen in order to best discriminate the colorsof basophils, monocytes, lymphocytes (all shades of blue), eosinophils(red), and neutrophils (neutral color). For counting platelets, forexample, two colors of illumination may be used (such as 430 nm and 570nm). A high contrast image may be obtained by subtracting the 430 nmimage from the 570 nm image. Light having a wavelength of 430, 500, 525and 600 are particularly effective at showing cell color information,but light at wavelengths between 400 nm and 700 nm inclusive may beused. These wavelengths will also be used for the display of the colorimages if appropriate. Otherwise one or two additional images may needto be taken for the 200+ cells that will be analyzed for thedifferential count and which may be shown on the display 320. Typicallythe narrow-band images will be chosen from the range of 400 nm to 750nm. Test results have shown that 2-8 separate light colors to work well,with 3-4 separate light colors being optimal. The computer 300 may beable to further refine the images by compensating for spatial shifts.Also the computer may combine the various colored images to generatemulti color images for display or analysis. Numeric descriptors of theindividual images or combined images can be used to determine spatial,densitometric, colorimetric and texture features of the cells forclassification of the cell types. A further advantage of using narrowband illumination is that using narrow band illumination allows for theelimination of the use of oil objectives or coverslips. Light isrefracted when the light passes from glass to air. Prior art systemshave used oil objectives or coverslips to minimize this refraction atair to glass transitions, but having to add oil or coverslips adds stepsto processing the slides, and increases the per slide analysis cost. Toovercome, the need to use coverslips or oil, a combination of narrowband LEDS or filtered light can be used. Reducing the variance orbandwidth in the wavelengths of the light decreases the distortion inthe image captured by the light receiving device 200 when the lightpasses through the slide 701. The computer 300 may also instruct thelight emission device 600, to focus the light from the light source(either 610 or 630) so that the light is properly focuses on the slide.To do this, the computer 300 may instruct a focus adjustor to optimizethe focus for each color of light.

The Slide Apparatus 700

FIGS. 1A, 2, and 3 illustrate an embodiment of the slide apparatus 700comprising a slide 701, a specimen zone 710, a slide frame 720, and aslide holder 730. However, other embodiments of the invention may notrequire the use of a slide holder 730 or slide frame 720. Additionallythe specimen zone 710 boundary mark is optional as well, and maycomprise one or more hydrophobic rings or other painted marks. Theserings may help contain the blood sample, and also make reviewing imagesof the slides easier by quickly locating the specimen zone when a slideis viewed manually under a microscope (the may also assist the analysisprocess in interpreting the image.) The rings may also assist infacilitating the transfer of the stain onto the slides. Additionally,while the specimen zone has been illustrated as a rectangle other shapessuch as a circle or triangle may be used. Different size specimen zonesmay be used, including zones having a total area of one half to threesquare centimeters. The slide 701 may be manufactured from glass orplastic and may be 1 inch tall by 3 inches wide by 1 mm thick. Alsoshown on FIGS. 2 and 3 is a fluid sample dispersed on the slide in flows750, The fluid can be dispersed in flows as shown in FIG. 3, or in aspiral pattern as shown in FIG. 4.

The Discharge Device 900

With reference to FIG. 1B, the system may comprise a discharge device900 for pretreating the slide 701. The discharge device may take theform of a corona discharge device. The discharge device 900 may cleanthe slide 701 by creating a high intensity heat to burn off smallparticles to clean the slide to create a hydrophilic surface.Electro-Technic Products, Sawicki PA makes a corona discharge devicecompatible with the present invention. To perform the pretreatment, thecomputer 300 would turn on the discharge device 900, and cause the slideapparatus controller 760 to move the slide in a spiral or raster motionfor about 15 seconds (though a range of 1-20 seconds could be used.) Thedischarge device may be set at an angle from the slide, or may bepositioned directly above the slide. Typically, the discharge device 900may be positioned approximately 10 to 20 mm above the slide.

The Slide Labeler 1000 and Slide Label Reader 1100

The system 10 may optionally include a slide labeler 1000 and optionallya slide label reader 1100. The slide label reader 1000 may situated onthe platform 100 near the feeder 102 as shown in FIGS. 1A and 1B or maybe free standing or attached to other components. Slide labeler 1000 mayplace a label on the slide. A label 770 may include items such asstickers, barcodes, RFID tags, EAS tags, or other type of markings onthe slide. FIG. 3 shows an exemplary slide having a UPC bar code labelon it, but other markings conventions may be used. Moreover, themarkings may be applied directly to the slide via paint or ink, or maythey may be stuck to the slide using a writing medium and an adhesive(like a sticker).

The system 10 may comprise a slide label reader 1100. Slide label reader1100 may read markings placed on the slide from the slide labeler 1000or by labelers external to the system. The slide label reader 1100 couldcomprise an interrogator, a bar code reader, or other optical device. Insome embodiments, the system 10 may be able to determine informationfrom the labels 770 without a slide label reader 1100 by using the lightreceiving device 200 to capture an image of the label 770. The computer300 or the light receiving device (if it contains a processor andmemory) could perform imaging processing on the image containing thelabel and determine the information about the label 770.

Bone Marrow

As discussed above, the present invention may be used to analyzeperipheral or whole blood. The invention can also be used, however, tostudy cells of various types of body fluids. For example, thepreparation methods and analysis techniques described here can also beapplied to bone marrow aspiration samples. Bone marrow samples have ahigher cellular density and contain many immature red and white bloodcell types that are seldom found in peripheral blood. The technique ofpreparing a thin layer of cells, staining with a Romanowsky stain andanalyzing with image analysis can be applied to bone marrow aspirates aswell, however more sophisticated image analysis may be needed todiscriminate the additional types of cells.

As with peripheral blood samples, bone marrow samples may be collectedinto a container with an anticoagulant. This anticoagulant may be EDTAor heparin. Additional diluting or preserving fluid may be added to thesample. In the instrument described here a bone marrow sample would beprepared by first agitating the sample to provide a thorough mixing. Dueto the uncertain cellular density of such samples one or more dilutionsmay be prepared and pipetted onto the slide or slides. In oneembodiment, a triple dilution process may be used to create threespecimens. A first specimen may be created by adding 2 parts diluent toone part bone marrow. The first specimen may then be ejected onto afirst portion of the specimen zone 710 of the slide 701. A secondspecimen may be created by adding four parts diluent to the bone marrow.The second specimen may then be ejected onto a second portion of thespecimen zone 710 of the slide 701. A third specimen may be created byadding eight parts of diluent to the marrow. The third may then beejected onto a third portion of the specimen zone 710 of the slide 701.

For the image analysis, a low magnification assessment of the cellulararea on the slide could choose the optimum one third for subsequentanalysis. Once the proper area of the slide is selected, 200+ bonemarrow cells would be measured to determine the differential count.

Reticulocytes

The system 10 may also count the number of reticulocytes in a bloodsample. Using a Romanowsky stain to mark RNA, the computer 300 can countthe number of reticulocytes present in the specimen. When a Romanowskystain is used, the reticulocytes appear slightly bluer than other redblood cells, and are usually slightly larger. The computer 300 can useits analysis process (16A or 17B, of FIGS. 7A and 7B) to quantify theblue component of the red cells. The analysis process could measureintegrated optical density of a cell's difference image created bysubtracting one image taken with blue light of 430 nm (range of 400 to470 nm) from an image taken with non-blue light of 600 nm (range of 470to 750 nm). The analysis process could correlate the number of red bloodcells with a defined range of integrated blue component to a number ofreticulocytes counted manually or by flow methods using special stains.The accuracy of the analysis process can be further improved byrequiring the analysis process (16A or 17B) to measure the size, shape,color, and measured characteristics of cellular objects. For example,the analysis process could detect the difference between a red bloodcell with a bluish platelet lying under or over a red blood cell asopposed to a true reticulocyte.

Process Flows

Embodiments of the present invention are contemplated to processmultiple slide apparatuses 700 in a pipelined series as shown in FIG. 1Aor 1B, but embodiments which process the slide apparatuses 700 inparallel may also be constructed. Embodiments may be constructed whichcan process a large number (e.g. 10-20) of slide apparatuses in seriesor in parallel, or smaller volume systems 10 can be constructed(processing 4-8 slides at a time.) The following two paragraphs describean example process flow for FIGS. 1A and 1B, but alternate process flowsare possible and feasible through alternate embodiments of theinvention. These process flows are also illustrated in FIGS. 7A and 7B.Additionally, other configurations of the system are possible, and wouldlikely have different process flows. Moreover, although the steps arepresented in a series, many of the steps may be presented in a differentorder or performed simultaneously. Finally, most of the following stepsare optional, and may be removed from the process flow.

In the embodiment shown in FIGS. 1A and 7A, the software stored in thememory of the computer 300 may cause the computer to control the order,speed, and variables associated with processes 1A-16A. The process maybegin with computer 300 sending an instruction to the slide labeler 1000to place a label 770 on the slide 701. The labeling process 1A, may beperformed in the feeder 102 or may be performed on the ramp 104 or atthe slide apparatus controller 760. To move the slide apparatus 700 fromthe feeder 102, the computer 300 may send an instruction to the feeder102 to activate the feeder propulsion mechanism 103. The computer 300may also cause a feeder process 2A to begin which may include moving theslide apparatus 700 onto the advancer 110. The feeder process mayinclude utilizing the sensor to determine how many slides are in thefeeder 102. The computer may cause the advancer 110 to initiate anadvancing process 3A including moving the slide to the applicationstation A, and onto the slide apparatus controller 760 (if one ispresent). Once the slide apparatus is on the slide apparatus controller,the slide may be pretreated by the discharge device 900. Thepretreatment process 4A may include the slide controller 760 rotating orspinning the slide apparatus 700 as the discharge device burns thedebris from the slide 701. Once the optional pretreatment process 4A iscompleted the applicator process 5A may begin. The applicator process 5Amay comprise having an operator fill the reservoir tank 420 with diluentand reservoir tank 430 with body fluid. Body fluids such as urine,vaginal tissue, epithelial tissue, tumors, semen, spittle, peripheralblood, bone marrow aspirate or other body fluids may be used.Alternatively, the fluids may be aspirated automatically from apatient's sample vial. The mixer 440 may begin the mixing process 6A tomix the diluent with the body fluid in a certain ratio such as 2:1 (bodyfluid:diluent) to form a diluted body fluid. To apply the diluted bodyfluid to the slide 701, one of two body fluid application processes 7Aor 7B (FIGS. 7A and 7B, described above in conjunction with theapplicator 400) may be performed (but either process could be used forboth embodiments). After the application completes, the advancer 110 maycontinue the advancing process moving the slide apparatus to a secondstation B. Once the body fluid application process 7A is completed, thedrying process 8A may begin. The drying process may include using thegas movement device 500 to direct gas onto the slide for a period oftime (such as 20-30 seconds). Once the slide is dried, the body fluidmay be fixed using the fixation process 9A. After the body fluid isfixed, it may be stained using the staining process 7A. After the bodyfluid is stained, the excess stain may be removed using a stain removingprocess 11A. The stain removing process 11A may include a slide tiltingprocess wherein the slide is tilted at least partially in order to allowthe stain and or fixative to drain off the slide. To capture images ofthe specimen, the advancer 110 may continue advancing the slideapparatus to the imaging station C. At the imaging station C, the systemmay activate specimen illuminating process 12A and an imaging process15A, which uses the light emission device 600 and light receiving device200 respectively to illuminate the specimen and to capture images of theilluminated specimen. The computer 300 may direct the light emissiondevice to apply to different filters to the light to change thewavelength of emitted light using the light filtration process 13A.Alternatively, the LED illumination process of 13B may emit one or morewavelengths of light if a light emission device 600 comprises one ormore LEDs. A slide movement process 14A may be performed by the slidemover 201 to position the slide 701 in various X, Y, Z directionalpositions. Since in many embodiments, the magnification of the lens ofthe light receiving device will generate a view field that only containsa part of the total area of the specimen, the slide movement process 14Amay be utilized to move the specimen into different X, Y positionsallowing the light receiving device 200 to take multiple images 331 tocapture the entire specimen. The slide mover may also be able to movethe slide to multiple imaging stations allow light receiving devices totake images at various magnifications. The slide mover may also be ableto move the slide in the Z direction allowing one or more lightreceiving devices to take images at various magnifications, focusposition and light wavelengths. The system 10 may use the label reader1100 to read the labels on the slides (using the label reading process15A), or alternatively the computer may recognize symbols on the labelusing image recognition software. The light receiving device maytransfer the images to the computer through link 11. The computer maysave the images in internal memory and use its software to analyze theimages (using the analysis process 16A) to count the cells and performcalculations on the resulting data. The software may generate resultsincluding tables, charts, or a graph of the results 332, and may displaythe images 331 on the display 320 of the computer 300.

A second process flow is shown in FIG. 7B (also refer to FIG. 1B). Theprocess may begin with computer 300 sending an instruction to the slidelabeler 1000 to place a label 770 on the slide 701. The labeling process1B may be performed in the feeder 102 or may be performed on the ramp104 or at the slide apparatus controller 760. To move the slideapparatus 700 from the feeder 102, the computer 300 may send aninstruction to the feeder 102 to activate the feeder propulsionmechanism 103. The computer 300 may also cause a feeder process 2B tobegin which may include moving the slide apparatus 700 down the ramp 104onto the advancer 110. The feeder process 2B may include utilizing thesensor to determine how many slides are in the feeder 102. The computermay cause the advancer 110 to initiate an advancing process 3B includingmoving the slide to the application station A, and onto the slideapparatus controller 760 (if one is present). Once the slide apparatusis on the slide apparatus controller 760, the slide 701 may bepretreated by the discharge device 900. The pretreatment process 4B mayinclude the slide controller 760 rotating or spinning the slideapparatus 700 as the discharge device burns off any debris on the slide701. Once the optional pretreatment process 4B is completed theapplicator process 5B may begin. The applicator process 5B may comprisehaving an operator fill the reservoir tank 420 with diluent andreservoir tank 430 with body fluid. Alternatively, the fluids may beaspirated automatically from a patient's sample vial. Body fluids suchas peripheral blood or bone marrow aspirate may be used. The applicator400 may contain a third reservoir for containing the stain, and perhapsa fourth reservoir for containing fixative (however, in otherembodiments the stain and fixative could be stored in the samereservoir). The mixer 440 may begin the mixing process 6B to mix thediluent with the body fluid (and possibly the stain and fixative) in acertain ratio such as 2:1 (body fluid:diluent) to form a diluted bodyfluid. In these embodiments, the applicator 400 would apply the stainand or the fixative after the body fluid is applied to the slide usingthe staining process and fixative process respectively. Once the slideis dried, the body fluid may be fixated using the fixation process 9B.After the body fluid is fixed, it may be stained using the stainingprocess 7B. To apply the diluted body fluid to the slide 701, one of twobody fluid application processes 7A or 7B (described above inconjunction with the applicator 400) may be performed (but eitherprocess could be used for both embodiments.) Once the body fluidapplication process 7B is completed, the drying process 8B may begin.The drying process may include using the gas movement device 500 todirect gas onto the slide for a period of time (such as 20-30 seconds).After the body fluid is stained and fixed, the stain may be removingusing a stain removing process 11B. The stain removing process 11B mayinclude a slide tilting process wherein the slide is tilted at leastpartially in order to allow the stain and or fixative to drain off theslide. To capture images of the specimen, the advancer 110 may continueadvancing the slide apparatus to the imaging station C. At the imagingstation C, the system may activate specimen illuminating process 12B andan imaging process 15B, which uses the light emission device 600 andlight receiving device 200 respectively to illuminate the specimen andto capture images of the illuminated specimen. The computer 300 maydirect the light source 600 to apply a sequence of narrow band lightonto the slide 701 using LED illumination process 13B. Alternatively, ifa light emission device 600 with filters is provided, the computer 300may direct the light emission device to radiate light and applydifferent filters to the light to change wavelength of emitted lightusing a light filtration process 13A. Once slides are illuminated, aslide movement process 14B may be performed by the slide mover 201 toposition the slide 701 in various X, Y, Z positions. Since in manyembodiments, the magnification of the lens of the light receiving devicewill generate a view field which only contains a part of the total areaof the specimen, the slide movement process 14B may be utilized to movethe specimen into different X, Y positions allowing the light receivingdevice 200 to take multiple images to capture the entire specimen. Theslide mover may also be able to move the slide to multiple imagingstations allow light receiving devices to take images at variousmagnifications. The slide mover may also be able to move the slide inthe Z direction to allow the light receiving device to take images atvarious magnifications. The system 10 may use the label reader 1100 toread the labels on the slides (using the label reading process 16B), oralternatively the computer may recognize symbols on the label usingimage recognition software. The light receiving device may transfer theimages to the computer through link 11. The computer may save the imagesin internal memory and use its software to analyze the images (using theanalysis process 17B) to count the cells and perform calculations on theresulting data. The software may generate results including tables,charts, or graph of the results, and may display the images 331 or theresults 332 on the display 320 of the computer 300.

Test Results

To determine the accuracy of this method, computer algorithms weredeveloped to count RBCs and WBCs from digital images.

The image shown in FIG. 8 is of rows of a blood sample distributed on aslide generated by an automated applicator system. The fields at theedges of the square sample contain fewer cells as the applicator tipquickly turns to lay down the next row. At the end of the last row, a“blob” of cells was created where the applicator tip stopped and liftedfrom the surface of the slide. In such crowded fields of cells, thesoftware is still able to make an estimate of the red cell counts,although it appears that the computer counts may be slightly low incrowded areas. FIG. 9 shows the details of one digital image from theblood cell sample from which white blood cell and red blood cell countswere made.

An accurate count may be made by scanning the entire preparation for redblood cells and white blood cells, but an accurate platelet count mightuse a reduced number of fields, since an average cell deposition willhave a total of around 30,000 platelets, or approximately 500 plateletsper field. The image analysis programs then analyze each individualfield and sum the total red and white cell counts. To calculate thetotal counts per microliter in the patient vial, the number counted onthe slide is multiplied by the dilution factor.

Table 1 below shows a summary of data for 34 slides. “Invention” datarepresents red and white blood cell counts from slides produced usingthe method described above, and analyzed using image analysis countingalgorithms. “Sysmex” data represents red and white blood cell countsfrom a commercial “flow-based” automated CBC analyzer. Note that thespecimens include very high and very low red blood cell counts and whiteblood cell counts.

TABLE 1 Invention Sysmex Invention Sysmex Count Count Count CountSpecimen RBC × 10⁶ RBC × 10⁶ WBC × 10³ WBC × 10³ 1 4.97 5.69 5.00 5.64 23.66 4.22 5.92 6.99 3 4.32 4.83 4.13 4.00 4 4.00 4.01 3.36 2.91 5 4.274.22 9.66 8.48 6 2.83 3.20 8.60 9.25 7 4.46 4.79 5.80 6.40 8 4.04 4.784.02 4.63 9 2.98 3.10 10.02 10.16 10 4.88 5.04 6.24 6.44 11 2.95 3.297.28 8.43 12 4.47 4.97 6.75 7.70 13 2.75 3.01 4.91 4.62 14 4.35 4.738.48 9.27 15 3.82 4.16 6.26 6.06 16 3.16 3.50 14.49 14.97 17 3.87 4.225.37 4.67 18 3.69 4.04 3.75 3.50 19 4.08 4.51 11.42 11.22 20 3.03 3.262.00 1.87 21 3.23 3.49 6.68 6.50 22 4.35 4.63 10.09 9.95 23 2.84 3.0310.28 11.62 24 3.02 3.27 0.59 0.57 25 2.75 2.87 17.06 16.42 26 2.78 3.015.80 5.56 27 2.73 2.90 8.84 8.28 28 2.97 2.98 17.18 17.41 29 3.56 3.7516.70 16.79 30 2.91 3.16 7.05 7.89 31 3.32 3.55 9.80 9.73 32 3.01 3.2945.00 44.62 33 4.77 5.24 6.11 6.44 34 4.34 4.57 7.01 6.89 Table 1 showsthe raw data from counts performed on 34 vials. The second and thirdcolumns shows the red blood cell counts expressed as millions permicroliter of patient blood for the invention count and the Sysmexcount, respectively. The fourth and fifth columns shows the white bloodcell counts expressed as thousands per microliter of patient blood forthe invention count and the Sysmex count, respectively.

TABLE 2 Vial SysmexRBCs RBCcounts RBCscaled SysmexWBCs WBCcountsWBCscaled 1 5.69 1241765 4.97 5.64 1250 5.00 2 4.22 915262 3.66 6.991481 5.92 3 4.83 1080856 4.32 4.00 1033 4.13 4 4.01 998828 4.00 2.91 8403.36 5 4.22 1068411 4.27 8.48 2414 9.66 6 3.20 707250 2.83 9.25 21498.60 7 4.79 1115913 4.46 6.40 1451 5.80 8 4.78 1010933 4.04 4.63 10064.02 9 3.10 744241 2.98 10.16 2504 10.02 10 5.04 1220400 4.88 6.44 15596.24 11 3.29 736701 2.95 8.43 1819 7.28 12 4.97 1117506 4.47 7.70 16886.75 13 3.01 687645 2.75 4.62 1228 4.91 14 4.73 1086737 4.35 9.27 21208.48 15 4.16 955279 3.82 6.06 1564 6.26 16 3.50 789218 3.16 14.97 362214.49 17 4.22 967780 3.87 4.67 1343 5.37 18 4.04 922880 3.69 3.50 9373.75 19 4.51 1019878 4.08 11.22 2855 11.42 20 3.26 757606 3.03 1.87 5002.00 21 3.49 808679 3.23 6.50 1670 6.68 22 4.63 1086451 4.35 9.95 252210.09 23 3.03 709164 2.84 11.62 2571 10.28 24 3.27 753952 3.02 0.57 1470.59 25 2.87 688731 2.75 16.42 4265 17.06 26 3.01 695059 2.78 5.56 14515.80 27 2.90 682449 2.73 8.28 2209 8.84 28 2.98 741274 2.97 17.41 429517.18 29 3.75 890278 3.56 16.79 4174 16.70 30 3.16 727660 2.91 7.89 17627.05 31 3.55 831027 3.32 9.73 2450 9.80 32 3.29 753365 3.01 44.62 1125045.00 33 5.24 1193348 4.77 6.44 1527 6.11 34 4.57 1085941 4.34 6.89 17537.01 RBC Correlation (R{circumflex over ( )}2) 97.95% WBC Correlation(R{circumflex over ( )}2) 99.70% Table 2 shows the raw data from countsperformed on 34 vials. The 2^(nd) column gives the reference (Sysmex)RBC counts, while the 3^(rd) column reports the automated counts fromthe microscope slide. The 4^(th) column scales the counts to millioncells per microliter, assuming a 1:4 dilution. The 5^(th)-7^(th) columnshow the data for the WBC counts. At the bottom of the table are thecalculated correlation coefficients (R-squared).

The data was obtained from 34 patient samples during two sessions ofpreparing slides. The data is representative of typical patients,although the tubes were selected from patients with a wide distributionof red and white cell counts. Most, if not all of the 34 samples, wereobtained from specimens “archived” during the day in the refrigerator,and then pulled and prepared on the instrument in the late afternoon.Once the tubes were pulled, they were processed consecutively.

The algorithms were first validated by comparing manually countedmicroscope fields to the automated counts. There is a high correlationbetween the manually counted cells and the automatically counted cells.

High correlation between the two methods was found for both the redblood cell counts and the white blood cells counts (see Tables 1 and 2and FIGS. 5 and 6). The graph of FIG. 5 shows the correlation betweenthe Sysmex counts and the automated slide based counts for the red bloodcells. The data points are tightly clustered and form a line thatindicates that the numbers on the vertical axis (the invention counts)are similar to the numbers on the horizontal axis (the Sysmex counts).Typically for such data a correlation coefficient (R-squared) can becalculated to show the degree of agreement, where 100% would be perfectagreement. An R-squared value of 97.95% was calculated for this redblood cell data, indicating a high degree of agreement and similar towhat two different automated instruments might show. The graph shown inFIG. 6 shows the correlation between the Sysmex counts and the automatedslide counts for the white blood cells. The raw counts varied between147 and 11,250 white blood cells per slide. An R-squared value of 99.70%was calculated for this white blood cell data, indicating a high degreeof agreement and similar to what two different automated instrumentsmight show. This confirms that the novel approach to quantitativetransfer of cells was successful and that automated cell counts fromcomputer imaging yielded accurate results.

Exemplary Process for CBC and White Blood Cell (WBC) Differential

The following sequence of steps may be performed in any order and somesteps may be omitted or replaced with other steps.

-   Step 1. Extract a known volume of blood from a tube filled with a    patient's blood.-   Step 2. Dilute the blood if necessary. For example, one may use 5%    albumin in distilled water as a diluent.-   Step 3. Spread a known volume of blood or blood plus diluent over an    area on a glass microscope slide in a thin layer. The slide may be    treated to produce a hydrophilic surface to spread the cells better.    The slide may be treated to allow optimal adherence of the blood    elements to the slide.-   Step 4. Allow the slide to dry in the air, or assist the drying    using light air or heat.-   Step 5. Capture an image without a coverslip using a “dry” objective    that is corrected for no coverslip, for example one may use a 10× or    20× objective coupled to a CCD camera. Determine the count in each    image frame including Red Blood Cells (RBCs), and possibly White    Blood Cells (WBCs), and platelets. One or more colors may be used,    for example using a color camera or using narrow band illumination    produced by an interference filter or LED. Measurement of hemoglobin    content may be done at this time as well.-   Step 6. Fix and stain the cells on the slide. Fixation may be a    separate step or combined with staining.-   Step 7. Capture an image of stained slide without coverslipping,    using a “dry” objective, to count RBCs, WBCs, and platelets and    hemoglobin. This step may be in place of or in conjunction with step    5.-   Step 8. Perform WBC differential count from high resolution images    acquired without a coverslip, using a “dry” objective, for example    with a 40× or 50× objective that is not corrected for a coverslip. A    color camera or multiple black & white images taken using color    filters or using LED illumination may be used. This step may be in    addition to, or combined with step #7.-   Step 9, Calculate desired parameters and derived parameters required    for the CBC,-   Step 10. Display all CBC parameters to an operator in a Graphical    User Interface (GUI).-   Step 11. Display results of WBC differential to an operator in the    GUI.-   Step 12. Display images of RBCs, WBCs, platelets and any    unusual/abnormal blood elements to an operator.-   Step 13. Allow an operator to interact with the images and the    parameters to “sign off” the CBC, WBC differential count, and    identification of unusual or abnormal objects.-   Step 14. If needed, update results of CBC and WBC counts depending    on operator interaction in step #13.-   Step 15. Optionally, allow objects of interest to be relocated on a    microscope that has a motorized, computer controllable stage to    allow automated relocation of the objects for viewing.-   Step 16. Optionally, update the results of the CBC and WBC counts    depending on the microscopic operator interaction.

It is claimed:
 1. A system for analyzing cells from a blood sample,comprising: a. an applicator tip for dispensing a fluid comprising cellsfrom a blood sample onto a transparent substrate; wherein the tip isarranged above the transparent substrate; b. a light emitting diode(LED) light source arranged to emit light through the transparentsubstrate and through cells deposited on the transparent substrate,wherein the LED light source comprises two or more separate LEDs, eachLED emitting a different wavelength range; c. a light receiving devicearranged to capture one or more magnified black and white images of thecells on the transparent substrate; d. a display comprising a graphicaluser interface (“GUI”) arranged to display images of blood cells; e. acomputer containing a microprocessor; and f. software instructionsstored on a storage device for controlling the system, wherein when themicroprocessor executes the software instructions the computer causesthe system to: i. fill the applicator tip with a volume of the fluid;ii. position the applicator tip less than 70 microns above thetransparent substrate; iii. dispense a known volume of fluid out of theapplicator tip and while ejection of the fluid onto the transparentsubstrate is occurring, maintaining relative movement between the tipand the transparent substrate at a controlled speed to lay down theentire known volume of fluid in two or more rows over a defined area ofthe transparent substrate; and wherein the applicator tip height abovethe transparent substrate, a flow rate of the fluid out of theapplicator tip, and the speed of the relative movement are controlledsuch that cells in the fluid settle onto the transparent substrate in aplurality of rows that are about one cell thick, and such thatmorphology of the cells is sufficiently preserved to enable image-basedcell analysis; iv. control the LED light source to emit light throughthe transparent substrate and through the cells on the transparentsubstrate at two or more different wavelength ranges; v. activate thelight receiving device to capture a magnified black and white image ofone or more cells on the transparent substrate at each of the LED lightsource wavelength ranges; vi. generate a combined image of one or morecells on the transparent substrate by combining each of the black andwhite images; vii. automatically analyze red blood cells (RBCs) andwhite blood cells (WBCs) in the combined image and determine a RBC countper microliter of the blood sample, a complete blood count (CBC), and aWBC differential for the blood sample including a count of each type ofWBC per microliter of the blood sample; and viii. display the combinedimage and the results of the RBC count, CBC, the WBC differential, andthe count of each type of WBC in the GUI of the display, wherein the GUIis configured to allow an operator to interact with the combined imageand to sign off on the RBC count, the CBC, and the WBC differential. 2.The system of claim 1, wherein the applicator tip deposits a first rowof fluid along a first direction and turns at the end of the first rowto move in a second direction opposite to the first direction to laydown a subsequent row adjacent to the first row without lifting theapplicator tip away from the transparent substrate until after the knownvolume of fluid has been ejected out of the applicator tip.
 3. Thesystem of claim 1, wherein each LED of the two or more LEDs in the LEDlight source emits a different wavelength range selected from thefollowing wavelength ranges: 400+/−15 nm, 430+/−15 nm, 500+/−15 nm,525+/−15 nm, 570+/−15 nm, 600+/−15 nm, and 700+/−15 nm.
 4. The system ofclaim 3, wherein the LED light source comprises three or more separateLEDs, wherein each LED emits a different wavelength range selected fromthe following wavelength ranges: 430+/−15 nm, 525+/−15 nm, 570+/−15 nm,600+/−15 nm, and 700+/−15 nm.
 5. The system of claim 3, wherein the LEDlight source comprises three or more separate LEDs, wherein each LEDemits a different wavelength range selected from the followingwavelength ranges: 430+/−15 nm, 525+/−15 nm, and 600+/−15 nm.
 6. Thesystem of claim 3, wherein the LED light source comprises four or moreseparate LEDs, wherein each LED emits a different wavelength rangeselected from the following wavelength ranges: 430+/−15 nm, 525+/−15 nm,570+/−15 nm, 600+/−15 nm, and 700+/−15 nm.
 7. The system of claim 1,wherein the applicator tip comprises an orifice having an inner diameterof less than about 0.5 mm.
 8. The system of claim 7, wherein theapplicator tip orifice has an inner diameter of about 0.3 mm.
 9. Thesystem of claim 1, wherein the controlled speed of the relative movementbetween the tip and the transparent substrate comprises a speed of 10 to100 mm/second.
 10. The system of claim 1, wherein the magnification is10 to 20 times for use in determining the RBC count and CBC.
 11. Thesystem of claim 1, wherein the magnification is 40 to 50 times for usein determining the WBC differential.
 12. The system of claim 1, whereinthe transparent substrate comprises a slide.
 13. The system of claim 1,wherein the transparent substrate comprises a slide and the systemfurther comprises: a. a feeder for storing new slides; b. a firststation where the applicator applies the rows of fluid onto the slide;c. a second station for staining, fixing, and drying the slides, d. athird station for illuminating and imaging the slides; e. a collectorfor receiving processed slides; and f. an advancer for moving the slidesthrough the system starting from the feeder, moving the slides to afirst station, the second station, the third station; and then thecollector.
 14. The system of claim 1, wherein the computer causes thesystem to determine spatial, densitometric, colorimetric, and texturefeatures of the cells for classification of a cell type.
 15. The systemof claim 1, wherein the two or more rows are laid down adjacent to eachother on the transparent substrate and cells within adjacent rows settleadjacent one another to form a gapless or contiguous flow of cells inthe defined area.
 16. The system of claim 1, wherein the computer causesthe system to direct fluid out of the applicator tip at a flow rate ofabout 0.1 microliters per second.
 17. The system of claim 1, whereindisplayed combined images of the red blood cells include images ofreticulocytes and displayed results include a reticulocyte count. 18.The system of claim 1, further comprising a memory arranged to save theimages of blood cells.